The present invention relates generally isoforms of synapsin III, e.g., synapsin IIIa, IIIb, and IIIc. mRNA produced from transcription of the synapsin III gene undergoes alternative splicing and encodes the isoforms. Furthermore, the invention relates to uses of isoforms of synapsin III, and nucleic acids which encode these isoforms, especially in the diagnosis and treatment of schizophrenia.
The synapsins are a family of proteins that play a crucial role in the regulation of neurotransmission and in neurodevelopment. Single genes encode synapsins I and II. The synapsin I gene gives rise to two alternatively spliced mRNAs, which in turn, give rise to the synapsin Ia and Ib protein. The synapsin II gene gives rise to two alternatively spliced mRNAs, which in turn, give rise to the synapsin IIa and IIb protein (1).
The synapsin Ia, Ib, IIa, and IIb proteins are all found in the brain, where they are specifically localized to the presynaptic region of neurons, and coat synaptic vesicles. There is considerable experimental evidence that synapsins regulate the release of neurotransmission by becoming phosphorylated through the action of specific protein kinases (2).
There is also compelling evidence from several biological systems that synapsins play a critical role in axonal outgrowth, synapse formation, and synapse maintenance (3-8). Synapsins are potent stimulators of synapse formation, and may be useful as therapeutic agents for neurodegenerative diseases.
To date, no satisfactory candidate susceptibility gene for schizophrenia has been identified within the region of 22q 12-13 human chromosome 22, although several studies report that this region has a statistically significant chance of containing such a gene. Since synapsins play an integral role in neurotransmitter release and synaptogenesis, mutations in the gene therefor could explain many of the defects observed in neurotransmission and in synapse formation in schizophrenia.
Schizophrenia is a psychiatric illness that affects approximately 1% of the population worldwide. The illness is characterized by positive symptoms such as hallucinations, delusions, bizarre behavior, and thought disorder, as well as negative symptoms such as lack of motivation, social withdrawal, and apathy. Unfortunately, there is no objective laboratory test for schizophrenia, and the diagnosis is made by clinical interview. Current medication is effective for treating the positive symptoms of the disease, with little effect on the negative symptoms. Although the cause of schizophrenia is unknown, the disease has a strong genetic component. Research into the genetics of schizophrenia reveals that this disease is heterogeneous and is a xe2x80x9ccomplex geneticxe2x80x9d diseasexe2x80x94that is, several genes may be involved in the etiology of this disease. In identical twin studies, where one twin is affected by the disease, the other twin has a 50% chance of succumbing to the disease. Since identical twins possess identical genes, the penetrance of the disease in the setting of mutated genes is only 50%. Thus, an unknown environmental component as well as the presence of specific mutated genes, is required for the generation of schizophrenia.
Biochemical and cytological studies suggest that in schizophrenia, defects in neurotransmission and synapses occur. Abnormal neurotransmission affecting the dopamine, serotonin, glutamine, xcex3-aminobutyric acid, and cholecystokinin systems have been reported in schizophrenia (18). Neurodevelopmental abnormalities are also strongly implicated in schizophrenia, with reports of defects in neuronal cytoskeleton (19), neuronal cytoarchitecture and migration (20), cellular polarity (21), and synaptic pruning (22). Thus, in schizophrenia, at least two processes appear to be aberrant: neurotransmission and neuronal development, primarily affecting the later stages of synapse formation.
These studies indicate that a candidate susceptibility gene for schizophrenia should be expressed in the brain, and would likely play a role in synapse formation and neurotransmission. Furthermore, a candidate susceptibility gene should be genetically linked to schizophrenia. As indicated before, several research groups have independently identified a region on chromosome 22 that appears to possess a candidate susceptibility gene for schizophrenia (9-17). To date, a susceptibility gene has not been identified in this region.
Schizophrenia is a common disease, with a world-wide prevalence of 1%, affecting families of all races and socio-economic groups, and consuming a significant portion of all medical and social expenses.
Since there is a continuing need for new therapies for such diseases, efforts have been devoted to the characterization and elucidation of the genes for synapsins, and their various attendant uses. The susceptibility gene for schizophrenia has great commercial value both as a diagnostic reagent and for developing new treatments for this disease.
In accordance with the present invention, novel human cDNAs which encode isoforms of synapsin III, e.g., synapsin IIIa, IIIb, and IIIc have been cloned and characterized. These cDNAs are produced from alternative splicing of RNA complementary to the synapsin III gene, the newest member and third gene of the synapsin family of neuronal phosphoproteins.
This invention thus provides cDNAs of isolated clones that encode the heretofore unknown isoforms of the synapsin III protein, and which can be used to obtain genomic DNA, cDNA or RNA complementary to an isolated clone of the invention. Isolated clones of the invention can be used to isolate mRNA or genomic DNA from human, mammalian or other animal sources, or to isolate related cDNA or genomic clones by the screening of cDNA or genomic libraries. Furthermore, the clones themselves, or fragments derived from the clones, can be inserted in suitable vectors, such as plasmids or bacteriophages, then replicated and harvested following introduction into suitable bacterial host cells. DNA or RNA fragments derived from isolated clones of the invention can also be used as probes for in situ hybridization in order to locate tissues which express this gene, or for other hybridization assays for the presence of the gene or an mRNA transcriped therefrom in various biological tissues. In addition, oligonucleotides complementary to the sequence of an isolated clone of the invention can be synthesized and used as probes for this gene, an mRNA associated therewith, or for the isolation of related genes by homology screening of genomic or cDNA libraries, or by the use of amplification techniques such as the Polymerase Chain Reaction.
This invention may also be used to obtain isoforms of the synapsin III protein, or fragments thereof, produced from expression of an isolated genomic clone. For example, an isolated clone of the invention, or fragments thereof can be subcloned into suitable expression vectors, such as the plasmid pET15b (Novagen, Madison, Wis.), and transfected into suitable host cells, such as bacteria, yeast, or mammalian cells. As a result, an isoform of the synapsin III protein can be produced in the transfected host cell, and recovered from the host for direct uses or for experimental study, using methods well known in the art. Furthermore, pursuant to the invention, either an entire isoform the synapsin III protein or fragments thereof can be recovered from the transfected host for further uses.
This invention may also be used to generate antibodies directed against isoforms of human synapsin III protein. Production of an isoform, e.g., synapsin IIIa, IIIb, or IIIc, or fragments thereof, encoded by an isolated clone in transfected host cells such as those described above, would provide an isoform of the synapsin III protein, or fragments thereof, which could be used as antigen for the generation of polyclonal or monoclonal antibodies against isoforms of a synapsin III protein, using methods well known in the art. These antibodies could be used to detect the presence of an isoform of synapsin III protein in humans or animals, or in biological tissues or fluids isolated from humans or animals.
In addition, this invention may be used to isolate transcriptional or translational regulatory elements from the 5xe2x80x2 untranslated region of the synapsin III gene, or post-transcriptional regulatory elements from the 3xe2x80x2 untranslated regions of the isolated gene.
This invention can further be used to detect mutations in the synapsin III gene in individuals who are at risk of developing schizophrenia. Identification of these mutations will allow one to determine which individuals are at risk for developing schizophrenia.
Accordingly, it is a principal object of the present invention to provide various isoforms of the synapsin III protein and subunits or fragments thereof in purified form that exhibit certain characteristics and activities associated with synapsin III promotion of neurotransmitter activity and synaptogenesis.
It is a further object of the invention to provide isolated nucleic acid molecules which encode various isoforms of synapsin III, e.g., synapsin IIIa, IIIb, and IIIc. These isolated nucleic acid molecules are produced from the alternative splicing of RNA complementary to the synapsin III gene.
It is a further object of the invention to provide isolated nucleic acid molecules hybridizable under standard hybridization conditions to an isolated nucleic acid molecule that encodes an isoform of a synapsin III protein.
It is a further object of the present invention to provide antibodies to isoforms of synapsin III and their subunits, and methods for their preparation, including recombinant means.
It is a further object of the present invention to provide a method for detecting the presence of an isoform of synapsin III and its subunits in mammals in which invasive, spontaneous, or idiopathic pathological states due to neurotransmission deficiencies are suspected to be present, especially schizophrenia.
It is a further object of the present invention to provide a method and associated assay system for screening substances such as drugs, agents and the like, potentially effective in either mimicking the activity or combating the adverse effects of an isoform of synapsin III and/or its subunits in mammals.
It is a still further object of the present invention to provide a method for the treatment of mammals to control the amount or activity of isoforms of the synapsin III protein, or subunits thereof, so as to alter the adverse consequences of such presence or activity, or where beneficial, to enhance such activity.
It is a still further object of the present invention to provide a method for the treatment of mammals to control the amount or activity of isoforms of synapsin III or subunits or fragments thereof, so as to treat or avert the adverse consequences of invasive, spontaneous or idiopathic pathological states, especially schizophrenia and other neurodegenerative diseases.
It is a still further object of the present invention to provide pharmaceutical compositions for use in therapeutic methods which comprise or are based upon isoforms of synapsin III, or subunits thereof, their binding partner(s), or upon agents or drugs that control the production, or that mimic or antagonize the activities of isoforms of synapsin III, i.e., synapsin IIIa, synapsin IIIb, or synapsin IIIc.